Project Summary The reprogramming of skin or other easy to obtain cell types into therapeutically useful cells holds great promise for regenerative medicine, but the practical usefulness of this technology has been limited because typically only a small percentage of cells are successfully converted to the target cell type and these are often immature or incompletely specified. Single-cell expression analysis has revealed that most reprogrammed cells start with very similar expression profiles but then acquire a wide variety of different fates, many of which are developmental dead ends. Why does a cell population that is initially homogenous produce a myriad of different cell fates, with only a few cells achieving the targeted fate? This question is extremely difficult to answer with existing methods. The main focus of our proposal is to develop self-reporting Calling Cards, a new technology that can answer this question by simultaneously measuring transcription factor binding and genome-wide mRNA levels from thousands of single cells. We will demonstrate the utility of self-reporting Calling Cards by mapping the binding and function of the pioneer factor Foxa1 and its cofactor Hnf4a during the reprogramming of fibroblasts into induced endoderm progenitors (iEPs). We hypothesize that the direct targets of these TFs are stochastically expressed in cells undergoing lineage reprogramming, and that by forcing the expression of target genes that are usually transcribed only in successfully converted cells, we can improve overall target cell yield and maturity.